Unraveling the Mechanism of Cryptic Sexual Cycle Development in Amoeba, Cochliopodium, using Comparative Transcriptome and Immunocytochemistry The proposed work aims to unravel the cytological and genetic underlying of the cryptic sexual life cycle of an amoeboid lineage, Cochliopodium. Sexual reproduction is highly beneficial in eukaryotes by enabling them to increase: genetic diversity, fitness under selective pressure and virulence as in pathogenic eukaryotes. However, sexuality in most microbial eukaryotes is poorly understood. This is mainly due to their diverse quality and complex life cycle. Unlike most other amoebae including E. histolytica that are assumed to engage in sex during the dormant, cyst stage (a challenge for experimental study), Cochliopodium undergoes nuclear fusion in the actively growing vegetative stage. This makes it an ideal candidate for investigative work involving live experimentation. The proposed work will test three interrelated hypotheses including genetic mechanisms of sexual development, mechanisms of genome reduction and genetic recombination and mating system in Cochliopodium. We will use a combination of methods including comparative transcriptome obtained using next generation sequencing, cytological evidence on targeted sex genes using immunocytochemistry (ICC), and a series of culturing experimentation to understand mating system in Cochliopodium. We have already built bioinformatics pipeline and custom scripts to assemble and analyze the genetic data. Newly available statistical tools for de novo data will be used for comparative gene expression analysis. Addressing the proposed hypotheses will enable: (a) uncover cytological signatures of the stages of sexual development in Cochliopodium, (b) shed light on the molecular mechanisms of a possible cryptic sexual life cycle in Cochliopodium and others, (c) understand mechanisms of genome reduction and genetic recombination in cryptic life cycles, (d) exploration of targets that might be used to combat eukaryotic pathogens, and (e) provide insights into the origin and evolution of sexuality in microbial eukaryotes.